Molecular mechanisms of estrogen receptor β-induced apoptosis and autophagy in tumors: implication for treating osteosarcoma.

Estrogen receptors are ligand-dependent receptors that are located in the nucleus and composed of two subtypes: estrogen receptor α (ERα) and estrogen receptor β (ERβ). These two subtypes have similar structures, and both consist of a DNA binding domain, a ligand binding domain, a ligand-independent transcriptional activation region (AF-1) at the N-terminus, and a ligand-dependent transcriptional activation region at the C-terminus (AF-2). 1 Both estrogen receptor subtypes can initiate transcription of estrogen-responsive genes either in the form of a homodimer or a heterodimer after binding to estrogen. While the transcriptional activation function of AF-1 from ERβ is weaker compared with ERα, AF-2 from ERβ has comparable function to ERα. 2 Additionally, ERβ contains a repressor domain at the N-terminus. When hormones are at sub-saturated levels, ERβ can inhibit the transcriptional activity of ERα, thereby reducing the sensitivity of cells to estrogen. 3 ERβ has been proven to be a tumor suppressor in many tumor types. In prostate cancer cells, ERβ promotes apoptosis and inhibits cell proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT). 4 , 5 In ovarian cancer cells, ERβ inhibits cell growth and potentiates the antitumor activity of chemotherapy drugs, including cisplatin and taxol. 6 , 7 ERβ overexpression inhibits the growth of ERα-expressing breast cancer cells and prevents the production of estrogen-induced breast cancer xenografts in nude mice. 8 , 9 ERβ can influence downstream cell cycle progression by initiating the transcription of cell cycle-related target genes. 10 , 11 For example, in breast cancer cells that endogenously express ERα, ERβ overexpression prevents proliferation by inhibiting cyclin D1 expression and activating p21 and p27 expression to induce G2 cell cycle arrest. 8 In malignant pleural mesothelioma cells, ERβ functions as a tumor suppressor and its activation sensitizes tumor cells to cisplatin. 12 Moreover, ERβ expression has been revealed to be regulated by the AKT1/SIRT1/FOXM1 axis, while activated ERβ can inhibit AKT1 signaling, thus demonstrating an inhibitory feedback loop for ERβ. 13 ERα and ERβ have been identified in both healthy human bone cells and osteosarcoma cells, 14 – 16 and ERα and ERβ are stably expressed at a 1:4 ratio in the osteosarcoma cell line U2-OS. 17 However, in the osteosarcoma cell line 143B, which has high metastatic ability, only ERβ expression was detected. 18 In recent years, estrogen and its nuclear receptors have attracted widespread attention as potential targets for treating osteosarcoma. In the highly metastatic osteosarcoma cell line 143B, inhibition of cell proliferation by the 17-e-diol derivative 2-ME was more prominent when higher doses were used, and the estrogen inhibitor fulvestrant inhibited cell growth at high concentrations. Intriguingly, fulvestrant down-regulated ERβ expression, while 2-ME enhanced ERβ expression. 18 Therefore, the specific molecular mechanisms by which ERβ inhibits tumorigenesis in osteosarcoma cells are unclear. Recently, we reported that ERβ exerts antitumor effects in osteosarcoma U2-OS cells that are reliant on the roles of ERβ in regulating integrin, IAP, and the Nuclear factor-κB (NF-kB)/BCL-2 and phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) signaling pathways. 19 Here, we review the molecular mechanisms by which ERβ induces apoptosis and autophagy in a variety of tumors to further delineate more specific molecular mechanisms underlying osteosarcoma tumorigenesis and pathogenesis, as this might help pave the way for targeting ERβ to treat osteosarcoma and reduce mortality rates.

Generally, cell death is predominantly induced by apoptosis and autophagy, but other processes like necrosis, aging, and karyokinesis also result in cell death. Studies have confirmed that ERβ plays a role in inducing apoptosis in various tissues. In estrogen-treated mouse mammary cells, ERα promotes cell proliferation, whereas ERβ inhibits cell growth and induces apoptosis. 20 In human prostate cancer cells, ERβ induces apoptosis by enhancing the transcription of FOXO3a, which in turn elevates p53 upregulated modulator of apoptosis (PUMA) expression. 21 In nude mice, the ERβ activator diosgenin can inhibit the growth of prostate cancer xenografts. 22 Autophagy is another mechanism by which cell death occurs. Autophagy is usually activated in cells under stress conditions. 23 During autophagy, redundant proteins and/or organelles that do not affect survival are phagocytosed in double- or multi-layered vesicles to form autophagosomes. Subsequently, lysosomes are fused with autophagosomes and release proteases to degrade the contents of the autophagosomes. 24 Autophagy is a double-edged sword, such that it can be both beneficial and detrimental. Under normal environmental conditions, healthy cells maintain basal cellular activities and prevent malignant transformation through autophagy. In contrast, during stress, such as hypoxia or starvation, tumor cells can be controlled by autophagy pathways and are more likely to survive than healthy cells. 25 This also applies to conditions related to cancer chemotherapy, in which tumor cells can evade anticancer drugs through autophagy and become drug resistant. 25 It has been reported that autophagy suppresses tumors in most breast, uterine, and prostate cancers. 26 Studies have shown that estrogen receptors also induce autophagy. For instance, in hormone-resistant breast cancer cells, ERβ agonists reduce Bcl-2 expression and activate autophagy. 27 In Hodgkin's lymphoma, ERβ activation induces autophagy, inhibits proliferation, and causes cell cycle arrest. 28 Recently, complex interactions between autophagy and apoptosis have been uncovered. Multiple stresses activate both autophagy and apoptosis, which share multiple upstream and downstream regulatory molecules, and thus can be mutually transformed. 29 In osteosarcoma cells, lignin DPT can simultaneously induce apoptosis and autophagy. DPT induces autophagy by inhibiting activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway and blocking tumor cell apoptosis. In contrast, the autophagy inhibitor 3-methyladeine (3-MA) reverses this effect and promotes apoptosis. 30 An in-depth study of the interactions between autophagy and apoptosis will reveal mechanisms underlying the pathogenesis of various diseases and tumorigenesis of various cancers, shedding new light on methods to treat these cancers and other diseases.

LC3 is a microtubule-associated protein that is constitutively expressed in mammalian tissues. During autophagy, LC3-I, the cytosolic form of LC3, binds to phosphatidylethanolamine to form LC3-II, which is transported to autophagosome membranes. 52 When autophagosomes are fused to lysosomes to form autophagosomes, LC3-II in autophagosomes is degraded. 52 Thus, the relative ratio of LC3-I/LC3-II expression can be used to monitor autophagy progression. 53 In Hodgkin's lymphoma cells that are treated with lysosomal protease inhibitors, the ERβ agonist DPN enhances LC3-II expression. This suggests that ERβ induces autophagy to promote autophagosome formation and causes LC3-II formation even when the lysis function of lysosomes is inhibited. 28 P62 is a cytoskeletal protein with a ubiquitin-binding domain that has been found to co-localize with ubiquitinated protein aggregates in many neuropathic and liver diseases. 54 LC3 and the GABA type A receptor-associated protein (GABARAP) family proteins recognize and bind to specific sequences in p62. During autophagy, p62 recognizes toxic cellular waste, which is engulfed by autophagosomes and degraded by lysosomes. 55 When autophagy is inhibited, p62 and ubiquitinated protein aggregates in the cell accumulate, and when autophagy is activated, p62 levels continuously decrease. 54 , 55 Thus, p62 is used as a marker to study autophagic flow in cells. In choriocarcinoma cells, reactive oxygen species regulate the transition of methotrexate-induced apoptosis to autophagy through the JNK/p62 pathway, which results in the resistance of choriocarcinoma to methotrexate. 56 ERα overexpression in breast cancer cells with endogenous ERα expression has been reported to enhance p62 expression and activate autophagy. 57 However, the role of ERβ and p62 in autophagy has not been reported in the literature so far, and more extensive investigations are needed to explore their possible link. The protein kinase mTOR is the primary regulator of autophagy. mTOR receives signals from various pathways, especially those related to the cellular energy state and the initiation or arrest of protein synthesis. 58 mTOR forms two complexes, mammalian target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2), which have different protein compositions. 59 The PI3K/AKT signaling pathway is a major upstream regulator of mTORC1 and is normally activated by cell growth factors to promote cell survival and inhibit apoptosis in various cell types. 60 , 61 mTORC2 is involved in AKT phosphorylation. 59 , 60 Activation of AKT can lead to the phosphorylation of BAD, 62 inactivation of caspase-9, 63 inhibition of the nuclear transfer of the transcription factor FKHRL1 (which regulates the transcription of cell death genes), 64 and enhancement of mTOR activity, 65 thereby inhibiting apoptosis. In the case of starvation or environmental stress, inhibition of mTOR activity leads to the activation of the autophagy-activated kinase ULK1 and autophagosome formation. 66 Additionally, immunosuppressive drugs and rapamycin, which inhibits mTOR, initiates autophagy and autophagosome formation. 66 Overall, activation of the PI3K/AKT/mTOR pathway increases cell viability and prevents cell death caused by excessive autophagy, while inhibition of mTORC1 activity induces autophagy to clean up toxic waste in cells. Meanwhile, mTORC2 regulates AKT activity to increase cell viability. 67 The PI3K/AKT/mTOR pathway is associated with the estrogen receptor signaling pathway, and the downstream target gene p70S6K of mTORC1 negatively regulates AKT and activates estrogen receptors through phosphorylation. Moreover, in breast cancer cells, p70S6K overexpression has been reported to activate estrogen receptors. 68 , 69 In ERα-positive breast cancer, arctigenin, a member of the Asteraceae family, has been shown to inhibit mTOR pathway activation, resulting in decreased ER expression and increased autophagic cell death. 69 Additionally, arctigenin has also been reported to function as a selective agonist of ERβ to restrict mTORC1 activation in T cell lines; 70 thus, it is plausible that agonist-mediated activation of ERβ can inhibit mTORC1 to induce autophagic cell death in tumors. Possible mechanisms of ERβ-induced apoptosis in tumor cells. Estrogen/ERβ signaling has been shown to regulate the apoptosis-related proteins IAP, BAX, caspase-3, and PARP, and to act on the NF-κB/BCL-2 signaling pathway to induce apoptosis in tumor cells. ERβ, estrogen receptor β; IAP, inhibitors of apoptosis proteins; PARP, Poly (ADP-ribose) polymerase 1. In mammalian cells, the protein kinase AMPK senses ATP levels, thereby sensing the cellular energy status. 24 , 71 When the ATP/AMP ratio in cells decreases, AMPK is activated by the upstream protein kinase LKB1. 71 Activated AMPK phosphorylates and activates the TSC1/2 complex, which inhibits mTOR activity via Rheb, thereby initiating autophagy. 72 Autophagy leads to the reuse of nutrients in cells, which enhances ATP production and restores a normal ATP/AMP ratio. 71 Conversely, the LKB1–AMPK pathway leads to the phosphorylation and activation of the cyclin-dependent kinase inhibitor p27kip1. 73 Under conditions of growth factor withdrawal and nutrient deprival, p27kip1 responds to stress by inducing autophagy to prevent cells from entering the apoptotic process, allowing survival. 73 Several studies have shown that estradiol activates AMPK by enhancing phosphorylation of the alpha catalytic subunit (AMPKα) of AMPK. 74 – 76 In breast cancer and cardiomyocytes, ERα directly binds to AMPKα, and both ERα and ERβ interact with LKB1, which is upstream of AMPK. 77 In castrated male mice, testosterone activates the expression of the autophagosome-forming marker ALP and induces TSC2 expression to activate AMPKα, while ERα is downregulated and ERβ expression is enhanced in muscle cells. 78 These findings demonstrate that ERβ induces autophagy by activating the AMPK pathway. Possible mechanisms of ERβ-induced autophagy in tumor cells. Estrogen/ERβ signaling regulates expression of the autophagy-associated markers LC3-I/LC-3II and p62, and is involved in autophagy induction in tumors cells by inhibiting the PI3K/AKT/mTOR pathway and activating the AMPK pathway. ERβ, estrogen receptor β; AMPK, 5' AMP-activated protein kinase; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; BCL-2, B-cell lymphoma 2.

ERβ exhibits antitumor effects in different tumor types. ERβ regulates the apoptosis-related proteins IAP, BAX, caspase-3, and PARP, and influences NF-κB/BCL-2 signaling to induce apoptosis ( Figure 1 ). ERβ is also involved in the induction of autophagy by inhibiting the PI3K/AKT/mTOR pathway and activating the AMPK pathway ( Figure 2 ). Our previous report revealed that ERβ exerts antitumor effects in osteosarcoma U2-OS cells through the NF-κB/BCL-2 and PI3K/AKT/mTOR pathways. 19 Considering the broad involvement of ERβ in the linked processes of apoptosis, autophagy, it is plausible that the critical role of ERβ in inhibiting the proliferation and metastasis of osteosarcoma cells is closely related to its regulation of apoptosis and autophagy.

ERα (estrogen receptors α); ERβ (estrogen receptors β); EMT (epithelial–mesenchymal transition); PI3K (phosphoinositide 3-kinase); mTOR (mammalian target of rapamycin); 3-MA (3-methyladeine); PUMA (p53 upregulated modulator of apoptosis); IAP (inhibitors of apoptosis proteins); X-IAP (X-linked inhibitor of apoptosis); NF-κB (Nuclear factor-κB); PARP (Poly (ADP-ribose) polymerase); NAD (nicotinamide adenine dinucleotide); GABARAP (GABA type A receptor-associated protein); BCL-2 (B cell lymphoma/leukemia-2); BAX (Bcl-2 associated X protein); AMPK (adenosine monophosphate-activated protein kinase)